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Over the last 10 years, evidence has accumulated that autoimmune Addison’s disease (AAD) is a heterogeneous disease. Residual adrenal function, characterised by persistent secretion of cortisol, other glucocorticoids and mineralocorticoids is present in around 30% of patients with established AAD, and appears commoner in men. This persistent steroidogenesis is present in some patients with AAD for more than 20 years, but it is commoner in people with shorter disease duration. The clinical significance of residual adrenal function is not fully clear at the moment, but as it signifies an intact adrenocortical stem cell population, it opens up the possibility of regeneration of adrenal steroidogenesis and improvement in adrenal failure for some patients.

Mesenchymal stem cells in the dental pulp exhibit a tendency for differentiation into various dental lineages and hold great potential as a major conduit for regenerative treatment in dentistry. Although they can be readily isolated from teeth, the exact characteristics of these stem cells have not been fully understood so far. When compared to twodimensional (2D) cultures, three-dimensional (3D) cultures have the advantage of enriching the stem cell population. Hence, 3D-organoid culture and 3D-sphere culture were applied to dental pulp cells in the current study. Although the establishment of the organoid culture proved unsuccessful, the 3D-sphere culture readily initiated the stable generation of cell aggregates, which continued to grow and could be passaged to the second round. Interestingly, a significant increase in SOX2 expression was detected in the 3D-spheroid culture compared to the 2D culture. These results indicate the enrichment of the stemness-high population in the 3D-sphere culture. Thus, 3D-sphere culture may act as a link between the conventional and 3D-organoid cultures and aid in understanding the characteristics of dental pulp stem cells.

Asymmetric cell division (ACD) of neural stem cells and progenitors not only renews the stem cell population but also ensures the normal development of the nervous system, producing various types of neurons with different shapes and functions in the brain. One major mechanism to achieve ACD is the asymmetric localization and uneven segregation of intracellular proteins and organelles into sibling cells. Recent studies have demonstrated that liquid-liquid phase separation (LLPS) provides a potential mechanism for the formation of membrane-less biomolecular condensates that are asymmetrically distributed on limited membrane regions. Moreover, mechanical forces have emerged as pivotal regulators of asymmetric neural stem cell division by generating sibling cell size asymmetry. In this review, we will summarize recent discoveries of ACD mechanisms driven by LLPS and mechanical forces.

In their seminal paper, Gans and Northcutt (1983) proposed that evolution of the vertebrate “New Head” was enabled by advent of the neural crest and cranial placodes(1). The neural crest is a stem cell population that arises adjacent to the forming central nervous system and contributes to important cell types including components of the peripheral nervous system, craniofacial skeleton, and elements of the cardiovascular system(2). In the past few years, the New Head hypothesis has been challenged by the discovery in invertebrate chordates of cells with some but not all characteristics of vertebrate neural crest cells(3–7). Here, we discuss recent findings regarding how neural crest cells may have evolved during the course of deuterostome evolution. The results suggest that there was progressive addition of cell types into the repertoire of neural crest derivatives throughout vertebrate evolution(8). Novel genomic tools have enabled higher resolution insight into neural crest evolution from both a cellular and gene regulatory perspective(9,10). Together, these data provide clues regarding the ancestral neural crest state and how the neural crest continues to evolve to contribute to the success of vertebrates as efficient predators.

Blood vessels induce epidermal proliferating clusters in the vicinity of activating stem cells to populate expanding skin., Stem cell (SC) proliferation and differentiation organize tissue homeostasis. However, how SCs regulate coordinate tissue scaling in dynamic organs remain unknown. Here, we delineate SC regulations in dynamic skin. We found that interfollicular epidermal SCs (IFESCs) shape basal epidermal proliferating clusters (EPCs) in expanding abdominal epidermis of pregnant mice and proliferating plantar epidermis. EPCs consist of IFESC-derived Tbx3+–basal cells (Tbx3+-BCs) and their neighboring cells where Adam8–extracellular signal–regulated kinase signaling is activated. Clonal lineage tracing revealed that Tbx3+-BC clones emerge in the abdominal epidermis during pregnancy, followed by differentiation after parturition. In the plantar epidermis, Tbx3+-BCs are sustained as long-lived SCs to maintain EPCs invariably. We showed that Tbx3+-BCs are vasculature-dependent IFESCs and identified mechanical stretch as an external cue for the vasculature-driven EPC formation. Our results uncover vasculature-mediated IFESC regulations, which explain how the epidermis adjusts its size in orchestration with dermal constituents in dynamic skin.

With the extensive increase in the senescence populace, the requirement for potent regenerative therapy approaches for the older is pivotal. Current signs of progress in our insight of resident stem cell population in different tissues of the adult body present possibility for the advancement of cell-based therapies to treat age-related diseases and trauma. However, clinical analysis and understanding from the model organisms have recognized considerable blockade that normal phenomenon of aging inflicts on the regeneration of tissue. An insight into the alterations to the stem cell microenvironment and the intrinsic milieu and the cell-intrinsic stem cell alterations are important for the advancement of regenerative therapy approaches. These new understandings recommend that particularly targeting the components of environmental niche that are distressed by the phenomenon of aging will be crucial for the development of new approaches to advance stem cell activity and progress the tissue repair approaches.

Modern single cell experiments have revealed unexpected heterogeneity in apparently functionally ‘pure’ cell populations. However, we are still lacking a conceptual framework to understand this heterogeneity. Here, we propose that cellular memories—changes in the molecular status of a cell in response to a stimulus, that modify the ability of the cell to respond to future stimuli—are an essential ingredient in any such theory. We illustrate this idea by considering a simple age-structured model of stem cell proliferation that takes account of mitotic memories. Using this model we argue that asynchronous mitosis generates heterogeneity that is central to stem cell population function. This model naturally explains why stem cell numbers increase through life, yet regenerative potency simultaneously declines.

Skeletal muscle is an ideal target for cell therapy. The use of its potent stem cell population in the form of autologous intramuscular transplantation represents a tantalizing strategy to slow the progression of congenital muscle diseases (such as Duchenne Muscular Dystrophy) or regenerate injured tissue following trauma. The syncytial nature of skeletal muscle uniquely permits the engraftment of stem/progenitor cells to contribute to new myonuclei and restore the expression of genes mutated in myopathies. Historically however, the implementation of this approach has been significantly limited by the inability to expand undifferentiated muscle stem cells (MuSCs) in culture whilst maintaining transplantation potential. This is crucial, as MuSC expansion and/or genetic manipulation is likely necessary for therapeutic applications. In this article, we review recent studies that have provided a number of important breakthroughs to tackle this problem. Progress towards this goal has been achieved by exploiting biochemical, biophysical and developmental paradigms to construct innovative in vitro strategies that are guiding stem cell therapies for muscle repair towards the clinic.

For prepubertal boys at risk of infertility, there are currently no established options for fertility preservation. The possibility for future fertility in these individuals is dependent on survival of the spermatogonial stem cell population during prepuberty testis. These stem cells will ultimately give rise to mature gametes following the onset of spermatogenesis at puberty. In this chapter we will describe the important changes that occur in the testis during prepuberty and the clinical situations in which fertility may be compromised during this period, with a particular focus on the effects of cancer treatment during childhood. We will discuss the present options for cryopreserving testicular tissue from prepubertal boys at risk of infertility and the experimental approaches that are under investigation as future clinical strategies for fertility restoration.

The adult mammalian heart was long thought to be a nonregenerating organ; however, recent evidence suggests otherwise. After many studies, a consensus has emerged that low levels of cardiomyocyte turnover occurs in the adult mammalian heart, originating primarily from preexisting cardiomyocytes with negligible contributions from any type of stem cell population. In this chapter, we highlight the critical studies that helped arrive at this conclusion We also discuss innovative approaches and potential therapeutic strategies that can generate new functional cardiac muscle tissue in the postnatal mammalian heart.

Milk is a complex fluid required for development, nutrition and immunological protection to the newborn offspring. Interestingly, latest finding proved the presence of novel stem cell population in human milk with multilineage differentiation potential. Given that little is known about cellular milk content in other mammalian species such as bovine, the purpose of our study was to isolate and characterize a potential stem cell-like population in bovine milk. In detail, we first analyzed the phenotype of the isolated cells able to grow in plastic adherence and then their capability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bovine milk stem cells (bMSCs) resulted plastic adherent and showed a heterogeneous population with epithelial and spindle-shaped cells. Successively, their immunophenotype indicated that bovine milk cells were positive for the typical epithelial markers E-cadherin, cytokeratin-14, cytokeratin-18, and smooth muscle actin. Notably, a subset (30%-40%), constantly observed in purified milk cells, showed the typical mesenchymal surface antigens CD90, CD73, and CD105. Furthermore, the same percentage of bMSCs expressing CD90, CD73, and CD105 presented the stemness markers SOX2 and OCT4 translocated in their nuclei. Finally, our data showed that bMSCs were able to differentiate into osteoblasts, chondroblasts, and adipocytes. In addition, the flow cytometry analysis revealed the presence of a subpopulation of events characterized by typical extracellular vesicles (EVs, size 0.1-1 μm), which did not contain nuclei and were positive for the same markers identified on the surface of bMSCs (CD73, CD90, and CD105), and thus might be considered milk cell-derived EVs. In conclusion, our data suggest that bovine milk is an easily available source of multipotent stem cells able to differentiate into multiple cell lineages. These features can open new possibilities for development biology and regenerative medicine in veterinary area to improving animal health.

Self-renewal is the process by which stem cells give rise to stem cells. Recent insights from experimental and theoretical models suggest that self-renewal not only influences the stem cell population but has a crucial impact on dynamics of non-stem cells. Efficient production of mature cells in tissue regeneration requires up-regulation of stem cell self-renewal. Increased self-renewal confers a competitive advantage on cancer cell clones by leading to aggressive expansion of both stem and non-stem cancer cells. Recent models suggest that self-renewal is the key parameter to understand clonal competition, selection and emergence of resistance in cancer cell populations.

Tissue development requires a control over the sequence of symmetric and asymmetric stem cell divisions to obtain the specific numbers of differentiated cells populating the tissue and stem cells residing in the niche. A good experimental model to study this process is the mouse intestinal crypt development, where it has been shown that stem cells follow an optimal strategy in which asymmetric division occurs only after all symmetric divisions have taken place to reach a fixed number of cells in the niche in the shortest time. Here we introduce a model of stem cell division that is able to explain the experimentally observed stem cell population dynamics by the effect of mechanical forces acting on the spindle. We also observe that the mechanically induced strategy for development is sub-optimal and crucially depends on the stiffness of the spindle. These findings highlight the crucial importance of mechanical forces for the development and maintenance of the intestinal crypt.

Hematopoietic stem cells (HSC) are one of the earliest, and most extensively studied stem cells that have been used in clinical medicine and serve as an excellent model in stem cell study. HSC are a heterogeneous population consisting of functionally different subsets. HSC transplantation is a golden standard for detection of self-renewal and multi-potential differentiation ability. Nowadays, population-based research cannot meet the need for deep understanding of biological functions and it is necessary to study at the single cell level. In this article, we introduce experimental procedures and data analysis of single cell transplantation. Researchers in related fields may find these technologies useful in their studies. As such, single cell biology is expected to increasingly expand for understanding of fundamental basis in medical science, understand applicability of single cell transplantation, acquire specific experimental operation, and promote wide applications, thus acting as a paradigm and reference for the study of other heterogeneous stem cell population.

Summary The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hopx+ colitis-associated regenerative stem cell (CARSC) population that functionally contributes to mucosal repair in mouse models of colitis. Hopx+ CARSCs, enriched for fetal-like markers, transiently arose from hypertrophic crypts known to facilitate regeneration. Importantly, we established a long-term, self-organizing two-dimensional (2D) epithelial monolayer system to model the regenerative properties and responses of Hopx+ CARSCs. This system can reenact the “homeostasis-injury-regeneration” cycles of epithelial alterations that occur in vivo. Using this system, we found that hypoxia and endoplasmic reticulum stress, insults commonly present in inflammatory bowel diseases, mediated the cyclic switch of cellular status in this process., Graphical Abstract, Highlights • Identify a regenerative stem cell population in colitis-associated regeneration • A long-term 2D culture to reenact the crypt homeostasis-injury-regeneration cycles • Oxygen tension serves as a switch in injury and regeneration in vivo and in vitro, An Lgr5+-independent population of stem cells maintains homeostasis in the colon and is associated with colitis-associated regeneration.

Heterogeneity within a putative stem cell population presents a challenge for studies and applications of such cells. Cloning may provide a strategy for reducing heterogeneity. However, previous studies have the weakness in reliability of single-cell-origin of the colonies. The present study aims to apply an alternative method to obtain clonal dental pulp cells with increased reliability of single cell origin. Dental pulp cells were cultured from 13 human wisdom teeth. Primary cultures of 3 human benign tumors were included as comparison. Cells were seeded into wells of a 96-plate at a mean density of 1 cell/well. On the next day, wells were inspected one by one to identify wells with single cells which were followed for 3 weeks. Survived clones were expanded and further characterized. Single cells were observed in all cases, the number of single-cell-wells varied from 16 to 33. Three weeks later, survived and grown clonal cells were observed in 10 to 29 wells, giving surviving rates of 33-91%. By contrast, though single tumor cells were also observed, none of them survived. Expanded clones exhibited diversity in viability and osteogenic differentiation which also differed from their parental cells. Seeding cells at clonal density into physically separated compartments like wells in a 96 plate and comprehensive observation provides a practical strategy for increasing reliability of single-cell origin of clonal cells. Cellular heterogeneity seems to be an intrinsic feature of dental pulp cells.

Summary Mechanical injuries cause widespread damage to tissues, fundamentally changing the physiological environment for subsequent repair. How these changes influence stem cells, which are central drivers of tissue repair and regeneration, is unknown. Planarian flatworms are highly capable regenerators, due to an abundant stem cell population that responds to wounding. Injury causes differentiated cells to die (Pellettieri et al., 2010) and stem cells to proliferate (Wenemoser and Reddien, 2010), but how these two events may be linked is unclear. To visualize stem cell dynamics after injury, we reduced their numbers by exposing animals to low doses of radiation (Wagner et al., 2011) and asked how the remaining stem cells respond to damage. We find that stem cells accumulate adjacent to injuries in large numbers, but only if injured within a defined window surrounding radiation. This accumulation occurs despite an absence of proliferation, indicating that stem cells are retained around the wound after radiation. Injury interferes with radiation-induced cell death, as stem cells from injured animals initiate apoptosis at significantly lower rates than uninjured controls. Injury is known to induce apoptosis in differentiated cells. By inducing rampant cell death throughout the body, we find that dying cells are sufficient to prolong survival of radiated stem cells. Together, our results suggest a model in which dying cells provide a protective signal to nearby stem cells, altering their susceptibility to radiation-induced apoptosis.